Study of extracellular ice-nucleating matter from ice-nucleating bacteria

通过超滤、蔗糖密度梯度超速离心法和凝胶过滤等方法，纯化了由冰核细菌Erwinia uredovora Kuin-3产生的胞外冰核物质(EM)。经透射电子显微镜观察，纯化的EM为球状物质(0.2-0.4um)。EM的冰核活性与该菌株细胞的冰核活性相当。从D_2O和H_2O冰冻素谱的差异判断，EM具有A类(-5ﾟC)、B类(-5-8ﾟC)和C类(-8ﾟC)结构。此外，经二胺氧化酶处理后，来自EM的A类和B类酶的活性降低。在25ﾟC以下的温度下，纯化的EM活性稳定，pH值在5.0~10之间。结果表明，EM的主要成分是脂肪(10%)、蛋白质(43%)、糖(35%)和多胺(12%)。加入抑制S-腺苷甲硫氨酸脱氧酶活性的乙二醛双(鸟苷酸){MGBG}，抑制该细菌细胞的冰核活性。经MGBG处理后，细胞和外膜中的多胺含量减少。结果表明，多胺是该细菌中最重要的成冰物质。

The extracellular ice-nucleating matter (EM) produced by the ice-nucleating bacterium, Erwinia uredovora KUIN-3, was purified to apparent homogeneity by ultrafiltration, sucrose density-gradientultracentrifugation, and gel filtration. The purified EM was sphercial matter (0.2-0.4mum) by examination using a transmission electron microscope. The ice nucleating activity from the EM was equal to that of the cells in this strain. It had become apparent that the EM had class A (--5ﾟC) , B ( -5--8ﾟC) , and C (-8ﾟC) structures as judged by its freezin differencespectrum in D_2O versus H_2O.The enzyme treatments by protease K,alpha-mannosidase, beta-galactosidase, and phospholipase C decreased the acyivity of the EM as well as of the cells. Further, the activities of class A and B from the EM were decreased by treatment with diamine oxidase. Also the activity of the purified EM was stable grom pH 5.0 to 10 and at temperatures below 25ﾟC.It was demonstrated that the components of the EM were lipid (10%) , protein (43%) , saccharide (35%) , and polyamine (12%) .The effects of polyamines on the ice-nuceating activity of an ice-nuceating bacterium, Erwinia uredovora KUIN-3, were investigated. The addition of methylglyoxal bis (guanylhydrazone) {MGBG} which inhibited the activity of S-adenosylmethionine decaboxylase, inhibited the ice-nucleating activity of the cell of this bacterium. The amounts of polyamines in the cells and outer membranes after MGBG treatment decreased. The results suggested that polyamine was the most important component of the ice-nucleating material in this bacterium.